1 Neuromodulators transform the output of neural circuits through their effects on diverse cellular processes and 2 play a particularly important role in the flexible control of behavior. Genetic manipulations of neuromodulators 3 in vivo leave no doubt that they exert a powerful influence on behavior. But it is difficult to pinpoint the role of a 4 given population of neuromodulatory neurons because their overall effect arises through their role on multiple 5 circuits in the brain and spinal cord. In particular, the specific role of neuromodulatory descending neurons 6 (NM-DNs)?neurons whose cell bodies reside in the brain and which send their axons to the spinal cord?in 7 modulating motor outputs is poorly understood. There is an urgent need to fill this gap in our knowledge 8 because NM-DNs are the major source of neuromodulators in the spinal cord and play a crucial role in shaping 9 motor output. The long-term aim of this research is to understand the role of NM-DNs in the context of 10 behaviors that require control over multi-jointed limbs. The overall objective in this proposal is to assess the 11 role of NM-DNs that use aminergic neurotransmitters?dopamine (DA), octopamine (OA) and serotonin 12 (5HT)?in a completely intact animal. The central hypothesis is that NM-DNs are recruited by DNs that mediate 13 sensorimotor transformations, which we refer to as sensory DNs (SDNs), and that SDNs initiate movement 14 while at the same time recruiting NM-DNs through their axon collaterals. NM-DNs do not initiate movement but 15 play a major role in descending motor control by modulating frequency, amplitude and duration of leg 16 movements. The rationale for this study is that a comprehensive understanding of descending 17 neuromodulation in an intact system in the context of multi-jointed limbs is essential to both the basic question 18 of neural control of complex behaviors, and to treatment strategies when such control is affected. The 19 proposed project has three specific aims: 1) To measure the relation between activities in NM-DNs and leg 20 kinematics. 2) To establish the circuit architecture underlying descending neuromodulation. 3) To understand 21 the effect of perturbing neuromodulation on a fly?s movement. Our approach is multidisciplinary: It employs a 22 combination of machine vision techniques to extract leg kinematics, a novel analytical framework for analyzing 23 leg movements, in vivo whole-cell patch clamp recordings to extract the relationship between activities in NM- 24 DNs and leg kinematics, and genetic tools to identify and perturb NM-DNs to establish a causal relationship 25 between NM-DNs and motor output. With regard to outcomes, we expect to elucidate how each of three 26 aminergic neurotransmitters functions individually and together in shaping motor output resulting from multiple 27 multi-jointed limbs. Such results are significant because they are expected to vertically advance understanding 28 of the role of NM-DNs because little is known about the circuit mechanisms by which they function in vivo. 29 Equally importantly, the results will have a positive effect on efforts to ameliorate the loss of motor control 30 during spinal injury by restoring neuromodulators.